This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: Cryo-EM single-particle reconstruction has evolved to become the most powerful approach to study ligand binding and conformational changes accompanying translation. A recent review (Frank, 2003) gives an overview over the many results that have accumulated since 1996, when tRNA binding to the ribosome was first visualized (Agrawal et al., 1996). EF-G mediated translocation was the first focus of the PI's research (Agrawal et al., 1998;1999;Frank and Agrawal, 2000), yielding three significant findings (i) EF-G binds to the ribosome in a position that is very similar to the binding position of the aminoacyl-tRNA-EF-Tu-GTP complex;(ii) EF-G undergoes a conformational change, characterized by a rotation of domains III-V;and (iii) the ribosome undergoes a "ratchet" motion, characterized by a rotation of the small subunit against the large subunit by as much as 10 degrees. Since the year 2000, we have collaborated with the group of Mans Ehrenberg in Uppsala, Sweden, enabling us to look at highly purified, kinetically characterized translational complexes in all different phases of translation. This collaboration has resulted in a number of major discoveries that have enriched our understanding of the molecular events. A partial list is given here briefly in telegraph style: (i) Decoding and tRNA accommodation are accompanied by a large change in tRNA conformation (Valle et al., 2002;2003a); (ii) The binding of a variety of factors to the ribosome leads to a characteristic conformational change of the stalk base (Rawat et al., 2003;Valle et al., 2003a); (iii) Binding of the tRNA D-loop to the L11-rRNA complex, observed by Valle and coworkers (2003a) is apparently facilitated by the flipping-out of a base of the rRNA (Li et al., submitted); (iv) The ratchet motion is a universal mechanism, observed with the binding of EF-G (Frank and Agrawal, 2000), EF2 to the 80S ribosome from yeast (Spahn et al., 2004), RF3 (U. Rawat et al., in preparation), and RRF (N. Gao et al., in preparation). (v) The ratchet motion involves an "elastic" deformation of the entire RNA matrix, and large movements and conformational changes of a number of ribosomal proteins (Gao et al., 2003); (vi) The ribosome can change from an "unlocked" state to a "locked" state, and this change is controlled by the state of acetylation of the P-site tRNA (Valle et al., 2003b); (vii) The L1 stalk is highly mobile, and its movement is anticorrelated with the movement of the small subunit head, such that the intersubunit space is opened and closed (Valle et al., 2003b). [While the movement involves a single hinge in the case of bacterial ribosomes (Valle et al., 2003), there are two hinges in the case of the eukaryotic ribosome (Spahn et al., 2004)]; (viii) When bound to the ribosome, release factors RF2 (Rawat et al., 2003) and RF1 (Rawat et al., in preparation) assume a conformation strongly different from that observed for RF2 by X-ray crystallography; (ix) RF3 has a conformation and binding position strikingly similar to those of EF-G (Rawat et al., in preparation). Using existing tools of cryo-EM single-particle reconstruction, and tools being developed in TRD2 and TRD3, we wish to pursue these studies with improved resolution, and follow the reaction pathways at increasing levels of detail. The ultimate goal is the description and understanding of molecular mechanisms underlying these processes. The experimental protocols for specimen preparation, electron microscopy, data processing, and interpretation have been described (Frank et al., 2000;Frank, 2002;Frank, 1996). Various time-resolved techniques outlined in TRD2 will be tried to capture additional states of the ribosome in the processes we have characterized. Resolution will be increased, by the use of automated data collection, improved image processing methods, and classification (TRD3), to improve the accuracy of docking and modeling of molecular interactions. Real-space refinement will be applied to determine the underlying molecular events, similar as has been done in Gao et al. (2003). Selected components of the ribosome will be studied by molecular mechanics simulations correlated with cryo-EM observations. References: 1. R.K. Agrawal, P. Penczek, R.A. Grassucci, Y. Li, A. Leith, K.H. Nierhaus, and J. Frank (1996) Direct visualization of A-, P-, and E-site transfer RNAs in the Escherichia coli ribosome. Science 271:1000-1002. 2. R.K. Agrawal, P. Penczek, R.A. Grassucci, and J. Frank (1998). Visualization of elongation factor G on the Escherichia coli ribosome: The mechanism of translocation. Proc. Natl. Acad. Sci. (USA) 95:6134-6138. 3. R.K. Agrawal, A.B. Heagle, P. Penczek, R.A. Grassucci, and J. Frank (1999) EF-G dependent GTP hydrolysis induces translocation accompanied by large conformational changes in the 70S ribosome. Nat. Struct. Biol. 6: 643-647. 4. J. Frank (1996) Three-dimensional Electron Microscopy of Macromolecular Complexes. Academic Press, San Diego. 5. J. Frank and R.K. Agrawal (2000) A ratchet-like inter-subunit reorganization of the ribosome during translocation. Nature, 406:318-322. 6. J. Frank, P. Penczek, R.K. Agrawal, R.A. Grassucci, and A.B. Heagle (2000) Three-dimensional cryoelectron microscopy of ribosomes. In Methods of Enzymology. Edited by D.W. Celander and J.N. Abelson, Academic Press, San Diego, CA. Chpt. 18, 276-291. 7. J. Frank (2003) Single-particle imaging of macromolecules by cryo-electron microscopy. Ann. Rev. Biophys. Biomol. Struct. 31:303-319. 8. J. Frank. (2003) Electron microscopy of functional ribosome complexes. Biopolymers 68: 223-233. 9. H. Gao, J. Sengupta, M. Valle, A. Korostelev, N. Eswar, S.M. Stagg, P. Van Roey, R.K. Agrawal, S.C. Harvey, A. Sali, M.S. Chapman, and J. Frank (2003) Study of the structural dynamics of the E. coli 70S ribosome using real space refinement. Cell 113:789-801. 10. H. Gao, M. Valle, M. Ehrenberg, and J. Frank (2004) Dynamics of EF-G interaction with the ribosome explored by classification of a heterogeneous cryo-EM dataset. J. Struct. Biol., in press. 11. U.B.S. Rawat, A.V. Zavialov, J. Sengupta, M. Valle, R.A. Grassucci, J. Linde, B. Vestergaard, M. Ehrenberg, and J. Frank. (2003) A cryo-electron microscopic study of ribosome-bound termination factor RF2, Nature 421:87-90. 12. C.M.T. Spahn, M.G. Gomez-Lorenzo, R.A. Grassucci, R. J[unreadable]rgensen, G.R. Andersen, R. Beckmann, P.A. Penczek, J.P.G. Ballesta, and J. Frank (2004) Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation. EMBO J., 23:1008-1019. 13. M. Valle, J. Sengupta, N.K. Swami, R.A. Grassucci, N. Burkhardt, K.H. Nierhaus, R.K. Agrawal, and J. Frank (2002) Cryo-EM reveals an active role for the aminoacyl-tRNA in the accomodation process. EMBL J., 21:3557-3567. 14. M. Valle, A. Zavialov, W. Li, S.M. Stagg, J. Sengupta, R.C. Nielsen, P. Nissen, S.C. Harvey, M. Ehrenberg, and J. Frank (2003a) Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-EM. Nature Struct. Biol., 10: 899-906. 15. M. Valle, A. Zavialov, J. Sengupta, U. Rawat, M. Ehrenberg, and J. Frank (2003b) Locking and unlocking of ribosomal motions. Cell, 114:123-134. The following papers, although not directly related to the RVBC, represent collaborations that benefited from the infrastructure provided by the RVBC: + Allen GS, Frank J (2007) Structural insights on the translation initiation complex: ghosts of a universal initiation complex. Molec Microbiol 63: 941-950. + Gillet R, Kaur S, Li W, Hallier M, Felden B, Frank J (2007) Scaffolding as an organizing principle in trans-translation: The roles of small protein B and ribosomal protein S1. J. Biol. Chem. 282, 6356-6363. + Mitra K, Frank J, Driessen A (2006) Co- and post-translational translocation through the protein-conducting channel: Analogous mechanisms at work? Nature Struct Mol Biol 13: 957-964. + Slagter-Jager JG, Allen GS, Smith D, Hahn IA, Frank J, Belfort M (2006) Visualization of a group II introns in the 23S rRNA of a stable ribosome. PNAS 103, 9838-9843. + Taylor DJ, Nilsson J, Merrill AR, Andersen GR, Nissen P, Frank J (2007) Structures of modified eEF2 + 80S ribosome complexes reveal the role of GTP hydrolysis in translocation. EMBO J. 26, 2421-2431.